Ligand functional substrates

ABSTRACT

A substrate comprising a crosslinked polymer primer layer, and grafted thereto a ligand-functionalized polymer is provided. The grafted polymer has the requisite affinity for binding neutral or negatively charged biomaterials, such as cells, cell debris, bacteria, spores, viruses, nucleic acids, and proteins, at pH&#39;s near or below the pI&#39;s of the biomaterials.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/468,302, filed Mar. 28, 2011, the disclosure of whichis incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present disclosure relates to ligand-functionalized substrates, andmethods for preparing the same. The functionalized substrates are usefulin selectively binding and removing biological materials, such asviruses, from biological samples.

BACKGROUND

Detection, quantification, isolation and purification of targetbiomaterials, such as viruses and biomacromolecules (includingconstituents or products of living cells, for example, proteins,carbohydrates, lipids, and nucleic acids) have long been objectives ofinvestigators. Detection and quantification are importantdiagnostically, for example, as indicators of various physiologicalconditions such as diseases. Isolation and purification ofbiomacromolecules are important for therapeutic uses and in biomedicalresearch. Biomacromolecules such as enzymes which are a special class ofproteins capable of catalyzing chemical reactions are also usefulindustrially; enzymes have been isolated, purified, and then utilizedfor the production of sweeteners, antibiotics, and a variety of organiccompounds such as ethanol, acetic acid, lysine, aspartic acid, andbiologically useful products such as antibodies and steroids.

In their native state in vivo, structures and corresponding biologicalactivities of these biomacromolecules are maintained generally withinfairly narrow ranges of pH and ionic strength. Consequently, anyseparation and purification operation must take such factors intoaccount in order for the resultant, processed biomacromolecule to havepotency.

The use of certain ionic polymers, especially cationic polymers, for theflocculation of cell and/or cell debris, as well as for theprecipitation of proteins, is known. Similarly, ionic polymers have beenused to modify filtration media to enhance the removal of impuritiesfrom process streams in depth filtration or membrane adsorber typeapplications. The effectiveness of these polymers is typically reducedas the conductivity of the media being processed increases, i.e. as thesalt content increases. There is a need in the art for polymericmaterials with increased affinity for biological species under highionic strength conditions.

Chromatographic separation and purification operations can be performedon biological product mixtures, based on the interchange of a solutebetween a moving phase, which can be a gas or liquid, and a stationaryphase. Separation of various solutes of the solution mixture isaccomplished because of varying binding interactions of each solute withthe stationary phase; stronger binding interactions generally result inlonger retention times when subjected to the dissociation ordisplacement effects of a mobile phase compared to solutes whichinteract less strongly and, in this fashion, separation and purificationcan be effected.

Most current capture or purification chromatography is done viaconventional column techniques. These techniques have severebottlenecking issues in downstream purification, as the throughput usingthis technology is low. Attempts to alleviate these issues includeincreasing the diameter of the chromatography column, but this in turncreates challenges due to difficulties of packing the columnseffectively and reproducibly. Larger column diameters also increase theoccurrence of problematic channeling. Also, in a conventionalchromatographic column, the adsorption operation is shut down when abreakthrough of the desired product above a specific level is detected.This causes the dynamic or effective capacity of the adsorption media tobe significantly less than the overall or static capacity. Thisreduction in effectiveness has severe economic consequences, given thehigh cost of some chromatographic resins.

Polymeric resins are widely used for the separation and purification ofvarious target compounds. For example, polymeric resins can be used topurify or separate a target compound based on the presence of an ionicgroup, based on the size of the target compound, based on a hydrophobicinteraction, based on an affinity interaction, or based on the formationof a covalent bond. There is a need in the art for polymeric substrateshaving enhanced affinity for viruses and other biological species toallow selective removal from a biological sample. There is further needin the art for ligand functionalized membranes that overcome limitationsin diffusion and binding, and that may be operated at high throughputand at lower pressure drops.

SUMMARY OF THE INVENTION

The present disclosure is directed to ligand-functionalized polymers,methods of making the same, and substrates bearing a grafted coating ofthe ligand-functional polymers. More specifically, the substratecomprises a crosslinked polymer primer layer, and grafted thereto aligand-functionalized polymer. The grafted polymer has the requisiteaffinity for binding neutral or negatively charged biomaterials, such ascells, cell debris, bacteria, spores, viruses, nucleic acids, andproteins, at pH's near or below the pI's of the biomaterials.

“Alkyl” means a linear or branched, cyclic or acyclic, saturatedmonovalent hydrocarbon having from one to about twelve carbon atoms,e.g., methyl, ethyl, 1-propyl, 2-propyl, pentyl, and the like.

“Alkylene” means a linear saturated divalent hydrocarbon having from oneto about twelve carbon atoms or a branched saturated divalenthydrocarbon having from three to about twelve carbon atoms, e.g.,methylene, ethylene, propylene, 2-methylpropylene, pentylene, hexylene,and the like.

“Alkenyl” means a linear unsaturated monovalent hydrocarbon having fromtwo to about twelve carbon atoms or a branched unsaturated hydrocarbonhaving from three to about twelve carbon atoms.

“Aryl” means a monovalent aromatic, such as phenyl, naphthyl and thelike.

“Arylene” means a polyvalent, aromatic, such as phenylene, naphthalene,and the like.

“Aralkylene” means a group defined above with an aryl group attached tothe alkylene, e.g., benzyl, 1-naphthylethyl, and the like.

“Heteroarylene” refers to a divalent group that is aromatic andheterocyclic. That is, the heteroarylene includes at least oneheteroatom in an aromatic ring having 5 or 6 members. Suitableheteroatoms are typically oxy, thio, or amino. The group can have one tofive rings that are connected, fused, or a combination thereof. At leastone ring is heteroaromatic and any other ring can be aromatic,non-aromatic, heterocyclic, carbocyclic, or a combination thereof. Insome embodiments, the heteroarylene has up to 5 rings, up to 4 rings, upto 3 rings, up to 2 rings, or one ring. Examples of heteroarylene groupsinclude, but are not limited to, triazine-diyl, pyridine-diyl,pyrimidine-diyl, pyridazine-diyl, and the like.

“hydrocarbyl” is inclusive of aryl and alkyl;

“(Hetero)hydrocarbyl” is inclusive of hydrocarbyl alkyl and aryl groups,and heterohydrocarbyl heteroalkyl and heteroaryl groups, the latercomprising one or more catenary (in-chain) heteroatoms such as ether oramino groups. Heterohydrocarbyl may optionally contain one or morecatenary (in-chain) functional groups including ester, amide, urea,urethane, and carbonate functional groups. Unless otherwise indicated,the non-polymeric (hetero)hydrocarbyl groups typically contain from 1 to60 carbon atoms. Some examples of such heterohydrocarbyls as used hereininclude, but are not limited to, methoxy, ethoxy, propoxy,4-diphenylaminobutyl, 2-(2′-phenoxyethoxy)ethyl, 3,6-dioxaheptyl,3,6-dioxahexyl-6-phenyl, in addition to those described for “alkyl”,“heteroalkyl”, “aryl”, and “heteroaryl” supra.

“(Hetero)arylene” is inclusive of arylene and heteroarylene.

DETAILED DESCRIPTION OF THE INVENTION

In the article and methods of this invention, ligand-functionalizedsubstrates are provided which have enhanced affinity and/or capacity,especially in high ionic strength media, for biological materials, suchas host cell proteins, DNA, RNA, viruses, and other microorganisms, atpH's near or below the pI's of the biological materials. The affinityfor such biomaterials allows materials that are positively charged atthose pH's, such as antibodies, to be purified, as they are not bound tothe ligand functional groups. The ligand functionalized substrate allowsthe selective capture or binding of target biomaterials by the ligandgroups, while other materials, lacking the affinity for the ligandgroups, are passed.

The ligand functional substrate comprises a) a substrate; b) a primerlayer disposed on the substrate comprising the reaction product of: 1) apolyamine polymer, 2) a polyfunctional crosslinking agent for thepolyamine polymer, and 3) an amine reactive monomer having apolymerizable, ethylenically unsaturated group, preferably a(meth)acryloyl group, and an amine-reactive functional group; and c) aligand-functional alkenyl (co)polymer layer grafted to the primer layer.The primer layer is coated on the substrate and cured to form a durable,crosslinked polyamine polymer layer having polymerizable, ethylenicallyunsaturated groups, preferably (meth)acryloyl groups, on the surfacethereof. The crosslinking of the polyamine polymer is effected by the 2)polyfunctional crosslinking agent, which has two or more amine-reactivefunctional groups, such as epoxy groups. The crosslinked polyaminepolymer is simultaneously or sequentially functionalized with primercomponent 3), having an amine-reactive group for coupling (by forming acovalent bond) to the crosslinked polyamine polymer, and anethylenically unsaturated group, such as a (meth)acryloyl group, whichmay be used to free-radically graft the c) ligand-functional alkenyl(co)polymer layer to the crosslinked polyamine polymer layer.

Polyamine Polymer

The primer base polymer comprises a polyamine polymer; i.e. a polymerhaving primary or secondary amino groups that may be pendent orcatenary, i.e. in the polymer chain. The aminopolymers contain primaryor secondary amine groups and can be prepared by chain growth or stepgrowth polymerization procedures with the corresponding monomers. Thesemonomers can also, if desired, be copolymerized with other monomers. Thepolymer can also be a synthesized or naturally occurring biopolymer. Ifany of these polymers, irrespective of source, do not contain primary orsecondary amine groups, these functional groups can be added by theappropriate chemistry.

Useful aminopolymers are water soluble or water-dispersible. As usedherein, the term “water soluble” refers to a material that can bedissolved in water. The solubility is typically at least about 1milligram, preferably 5 milligram, more preferably 10 milligram, permilliliter of water. As used herein, the term “water dispersible” refersto a material that is not water soluble but that can be emulsified orsuspended in water. In some embodiments mixed aqueous/alcoholic solventsystems may be advantageous.

Examples of aminopolymers suitable for use, which are prepared by chaingrowth polymerization include, but are not limited to: polyvinylamine,poly(N-methylvinylamine), polyethylenimine, polypropylenimine,polyallylamine, polyallylmethylamine, polydiallylamine,poly(-aminomethylstyrene), poly(4-aminostyrene),poly(acrylamide-co-methylaminopropylacrylamide), andpoly(acrylamide-co-aminoethylmethacrylate).

Examples of amino polymers suitable for use, which are prepared by stepgrowth polymerization include, but are not limited to: polyethylenimine,polypropylenimine, polylysine, polyornithine, polyaminoamides,polydimethylamine-epichlorohydrin-ethylenediamine, and certainpolyaminosiloxanes, which can be built from monomers such asaminopropyltriethoxysilane,N-(2-aminoethyl)-3-aminopropyltrimethoxysilane,N-trimethoxysilylpropyl-N-methylamine, andbis(trimethoxysilylpropyl)amine.

Useful aminopolymers may also include those that have primary orsecondary amino end groups and include, but are not limited to, thoseformed from polyamidoamine (PAMAM) and polypropylenimine: e.g. DAB-Amand PAMAM dendrimers (or hyperbranched polymers containing the aminefunctional group). Dendrimeric material formed from PAMAM arecommercially available under the trade designation Starburst™ (PAMAM)dendrimer (e.g., Generation 0 with 4 primary amino groups, Generation 1with 8 primary amino groups, Generation 2 with 16 primary amino groups,Generation 3 with 32 primary amino groups, and Generation 4 with 64primary amino groups) from Aldrich Chemical, Milwaukee, Wis. Dendrimericmaterial formed from polypropylenimine is commercially available underthe trade designation “DAB-AM” from Aldrich Chemical. For example,DAB-Am-4 is a generation 1 polypropylenimine tetraamine dendrimer with 4primary amino groups, DAB-Am-8 is a generation 2 polypropylenimineoctaamine dendrimer with 8 primary amino groups, DAB-Am-16 is ageneration 3 polypropylenimine hexadecaamine with 16 primary aminogroups, DAB-Am-32 is a generation 4 polypropylenimine dotriacontaaminedendrimer with 32 primary amino groups, and DAB-Am-64 is a generation 5polypropylenimine tetrahexacontaamine dendrimer with 64 primary aminogroups.

Examples of aminopolymers suitable for use, which are biopolymersinclude chitosan, glucosamine- and galactosamine-containingpolysaccharides, and starch, where the latter is reacted with reagentssuch as methylaminoethylchloride.

Other categories of aminopolymers suitable for use includepolyacrylamide homo- or copolymers with amino monomers includingaminoalkyl(meth)acrylate, (meth)acrylamidoalkylamine, and diallylamine.

Preferred aminopolymers include polyamidoamines, polyethyleneimine,polyvinylamine, polyallylamine, and polydiallylamine.

Suitable commercially available aminopolymers include, but are notlimited to, polyamidoamines such as ANQUAMINE™ 360, 401, 419, 456, and701 (Air Products and Chemicals, Allentown, Pa.); LUPASOL™polyethylenimine polymers such as FG, PR 8515, Waterfree, P, PS (BASFCorporation, Rensselaer, N.Y.); polyethylenimine polymers such asCORCAT™ P-600 (EIT Company, Lake Wylie, S.C.); and polyamide resins suchas the VERSAMID series of resins that are formed by reacting a dimerizedunsaturated fatty acid with polyalkylene polyamines (Cognis Corporation,Cincinnati, Ohio).

The primer layer has a crosslinking agent for the polyamine polymerhaving at least two amine-reactive functional groups, including ketone,aldehyde, ester, acyl halide, isocyanate, epoxide, anhydride, orazlactone groups. Preferably the amine-reactive functional groups Z areselected to react with the amine groups of the polyamine polymer attemperatures below about 50° C., preferably below 25° C. such that thecrosslinking reaction takes place during the coating and dryingoperation. Preferable crosslinking agents are further water-soluble orwater-dispersible.

Such crosslinking agents may have the general formula 1:R⁸—(Z)_(y),  1where R⁸ is a (hetero)hydrocarbyl group, Z is an amine-reactive groupand y is ≧2, preferably 2-4. The R⁸ group may be an alkylene group, anarylene group, a heteroarylene group, a heteroalkylene group, anaralkylene group, or a combination thereof.

In one embodiment the amine-reactive Z group may be an epoxy group andinclude both aliphatic and aromatic polyepoxides. Representativeexamples of aliphatic polyepoxides include3′,4′-epoxycyclohexylmethyl-3,4-epoxycyclohexanecarboxylate,3,4-epoxycyclohexyloxirane,2-(3′,4′-epoxycyclohexyl)-5,1″-spiro-3″,4″-epoxycyclohexane-1,3-dioxane,bis(3,4-epoxycyclohexylmethyl)adipate, the diglycidyl ester of linoleicdimer acid, 1,4-bis(2,3-epoxypropoxy)butane,4-(1,2-epoxyethyl)-1,2-epoxycyclohexane,2,2-bis(3,4-epoxycyclohexyl)propane, polyglycidyl ethers of aliphaticpolyols such as glycerol, ethylene glycol, polyethylene glycol orbutanediol. Representative examples of aromatic polyepoxides which canbe utilized in the composition of the invention include glycidyl estersof aromatic carboxylic acids, e.g., phthalic acid diglycidyl ester,isophthalic acid diglycidyl ester, trimellitic acid triglycidyl ester,and pyromellitic acid tetraglycidyl ester, and mixtures thereof;N-glycidylaminobenzenes, e.g., N,N-diglycidylbenzeneamine,bis(N,N-diglycidyl-4-aminophenyl)methane,1,3-bis(N,N-diglycidylamino)benzene, andN,N-diglycidyl-4-glycidyloxybenzeneamine, and mixtures thereof; and thepolyglycidyl derivatives of polyhydric phenols, e.g.,2,2-bis-[4-(2,3-epoxypropoxy)phenyl]propane, the polyglycidyl ethers ofpolyhydric phenols such as tetrakis(4-hydroxyphenyl)ethane,pyrocatechol, resorcinol, hydroquinone, 4,4′-dihydroxydiphenyl methane,4,4′ dihydroxydiphenyl dimethyl methane,4,4′-dihydroxy-3,3′-dimethyldiphenyl methane, 4,4′-dihydroxydiphenylmethyl methane, 4,4′-dihydroxydiphenyl cyclohexane,4,4′-dihydroxy-3,3′-dimethyldiphenyl propane, 4,4′-dihydroxydiphenylsulfone, and tris-(4-hydroxyphenyl)methane, polyglycidyl ethers ofnovolacs (reaction products of monohydric or polyhydric phenols withaldehydes in the presence of acid catalysts), and the derivativesdescribed in U.S. Pat. Nos. 3,018,262 and 3,298,998, the descriptions ofwhich are incorporated herein by reference, as well as the derivativesdescribed in the Handbook of Epoxy Resins by Lee and Neville,McGraw-Hill Book Co., New York (1967), and mixtures thereof.

In one embodiment the amine reactive functional group Z may be anisocyanate group. Suitable polyisocyanates include organic compoundscontaining at least two free isocyanate groups. Diisocyanates of theformula R⁸(NCO)₂ are preferably used wherein R⁸ denotes an aliphatichydrocarbon group with 4 to 20 carbon atoms, a cycloaliphatichydrocarbon group with 6 to 20 carbon atoms, an aromatic hydrocarbongroup with 6 to 20 carbon atoms or an araliphatic hydrocarbon group with7 to 20 carbon atoms.

Examples of diisocyanates include tetramethylene diisocyanate,hexamethylenediisocyanate (HDI), dodecamethylenediisocyanate,1,4-diisocyanatocy clohexane,1-isocyanato-3,3,5-trimethyl-5-isocyanatomethylcyclohexane (IPDI), 4,4′diisocyanato-dicyclohexylmethane (HMDI),4,4′-diisocyanato-2,2-dicyclohexyl-propane, 1,4-diisocyanatobenzene,2,4-diisocyanatotoluene (TDI), 2,6 diisocyanatotoluene,4,4′-diisocyanatodiphenylmethane (MDI), m- and p xylylenediisocyanate,α,α,α′,α′-tetramethyl-m- and p-xylylenediisocyanate and mixtures ofthese compounds. Suitable polyisocyanates also include triisocyanatessuch as 1,3,5 triisocyanatocyclohexane-s-trione, isocyanurate and biuretderivatives of HDI and MDI.

In one embodiment the amine reactive functional group Z may be anazlactone group. Reference may be made to Table 1 of a review entitled“Polyazlactones” by J. K. Rasmussen, et al., Encyclopedia of PolymerScience and Engineering, Second Edition, Volume 11, 1988, pp. 558-571that contains a listing of reported bis(azlactones). Other polyazlactone functional materials are described in U.S. Pat. No. 7,556,858(Rasmussen et al.), incorporated herein by reference.

In one embodiment the amine reactive functional group Z may be analdehyde or ketone group. Examples include bis- and polyaldehydes, suchas glyoxal or glutaraldehyde.

In some embodiments the crosslinking agent may be a polyacyl compoundwhere Z is an ester, acid, acid halide or anhydride group. Esters andacids are less preferred due to the reduced reactivity. Representativeexamples of suitable diacyl compounds, as the corresponding esters,halides, acids, and anhydrides: azelaic; maleic; fumaric; itaconic;1,5-pent-2-enedioic; adipic; 2-methyleneadipic; 3-methylitaconic;3,3-dimethylitaconic; sebacic; suberic; pimelic; succinic;benzylsuccinic; sulfosuccinic; glutaric; 2-methyleneglutaric;2-sulfoglutaric; 3-sulfoglutaric; diglycolic; dilactic;3,3′-(ethylenedioxy)dipropionic; dodecanedioic; 2-sulfododecanedioic;decanedioic; undecanedicarboxylic; hexadecanedicarboxylic; dimerizedfatty acids (such as those obtained by the dimerization of olefinicallyunsaturated monocarboxylic acids containing 16 to 20 carbon atoms, forexample, oleic acid and linoleic acid and the like); 1,2-, 1,4-, and1,6-cyclohexanedicarboxylic; norbornenedicarboxylic;bi-cyclooctanedicarboxylic; and other aliphatic, heteroaliphatic,saturated alicyclic, or saturated heteroalicyclic dicarboxylic acids;and the like; and mixtures thereof. Salts (for example, alkali metalsalts) of the above-described sulfonic acids can also be used.

The crosslinking agent for the polyamine polymer may be provided in anamount wherein the number of equivalents of amine reactive groups Z isat least 2%, preferably at least 5%, and up to about 20%, relative tothe number of equivalents of amine groups in the polyamine polymer.

The primer layer further comprises an amine reactive monomer having apolymerizable, ethylenically unsaturated group and an amine-reactivefunctional group, some embodiments of which are of the formula 2:

wherein

-   X¹ is —O— or —NR¹—, where R¹ is H or C₁-C₄ alkyl,-   R¹ is H or C₁-C₄ alkyl;-   R⁷ is a single bond or a (hetero)hydrocarbyl linking group,-   A is a functional group that is reactive with the amino groups of    the polyamine polymer, and-   x is 0 or 1.

In some embodiments compounds of Formula 2 are (meth)acryloyl compounds,and in other embodiments are alkenyl compounds.

Preferably, R⁷ is a single bond or a hydrocarbyl linking group thatjoins an ethylenically unsaturated, polymerizable group (e.g. alkenyl or(meth)acryl group) to reactive functional group A and preferably is analkylene group having 1 to 6 carbon atoms, a 5- or 6-memberedcycloalkylene group having 5 to 10 carbon atoms, or a divalent aromaticgroup having 6 to 16 carbon atoms; and A is a reactive functional groupcapable of reacting with an amine group of the polyamine polymer for theincorporation of a free-radically polymerizable group.

Useful reactive functional groups “A” include carboxyl, oxazolinyl,azlactone, acetyl, acetonyl, acetoacetyl, ester, isocyanato, epoxy,aziridinyl, acyl halide, and cyclic anhydride groups. Preferably theamine-reactive functional groups A are selected to react with the aminegroups of the polyamine polymer at temperatures below about 50° C.,preferably below 25° C. such that the reaction takes place during thecoating and drying operation. Preferable amine reactive monomers arefurther water-soluble or water-dispersible.

Representative azlactone group-substituted functional compounds ofFormula 2 include: 2-ethenyl-1,3-oxazolin-5-one;2-ethenyl-4-methyl-1,3-oxazolin-5-one; 2-isopropenyl-1,3-oxazolin-5-one;2-isopropenyl-4-methyl-1,3-oxazolin-5-one;2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one;2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one;2-ethenyl-4-methyl-4-ethyl-1,3-oxazolin-5-one;2-isopropenyl-3-oxa-1-aza[4.5]spirodec-1-ene-4-one;2-ethenyl-5,6-dihydro-4H-1,3-oxazin-6-one;2-ethenyl-4,5,6,7-tetrahydro-1,3-oxazepin-7-one;2-isopropenyl-5,6-dihydro-5,5-di(2-methylphenyl)-4H-1,3-oxazin-6-one;2-acryloyloxy-1,3-oxazolin-5-one;2-(2-acryloyloxy)ethyl-4,4-dimethyl-1,3-oxazolin-5-one;2-ethenyl-4,5-dihydro-6H-1,3-oxazin-6-one; and2-ethenyl-4,5-dihydro-4,4-dimethyl-6H-1,3-oxazin-6-one.

Representative acetoacetyl group-substituted functional compounds ofFormula 2 include 2-(acetoacetoxy)ethyl methacrylate.

Representative carboxyl group-substituted functional compounds ofFormula 2 include (meth)acrylic acid, 3-(meth)acryloyloxy-propionicacid, 4-(meth)acryloyloxy-butyric acid, 2-(meth)acryloyloxy-benzoicacid, 3-(meth)acryloyloxy-5-methyl benzoic acid,4-(meth)acryloyloxymethyl-benzoic acid, phthalic acidmono-[2-(meth)acryloyloxy-ethyl]ester, 2-butynoic acid, and 4-pentynoicacid.

Representative isocyanate group-substituted functional compounds ofFormula 2 include 2-isocyanatoethyl (meth)acrylate, 3-isocyanatopropyl(meth)acrylate, 4-isocyanatocyclohexyl (meth)acrylate,4-isocyanatostyrene, 2-methyl-2-propenoyl isocyanate,4-(2-(meth)acryloyloxyethoxycarbonylamino) phenylisocyanate, allyl2-isocyanatoethylether, and 3-isocyanato-1-propene.

Representative epoxy group-substituted functional compounds of Formula 2include glycidyl (meth)acrylate, thioglycidyl (meth)acrylate,3-(2,3-epoxypropoxy)phenyl (meth)acrylate,2-[4-(2,3-epoxypropoxy)phenyl]-2-(4-(meth)acryloyloxy-phenyl)propane,4-(2,3-epoxypropoxy)cyclohexyl (meth)acrylate, 2,3-epoxycyclohexyl(meth)acrylate, and 3,4-epoxycyclohexyl (meth)acrylate.

Representative acyl halide group-substituted functional compounds ofFormula 2 include (meth)acryloyl chloride, α-chloro(meth)acryloylchloride, (meth)acryloyloxyacetyl chloride, 5-hexenoyl chloride,2-(acryloyloxy) propionyl chloride, 3-(acryloylthioxy) propionoylchloride, and 3-(N-acryloyl-N-methylamino) propionoyl chloride.

Other useful amine-reactive monomers include anhydride group-substitutedfunctional monomers including maleic anhydride, (meth)acrylic anhydride,itaconic anhydride, 3-(meth)acryloyloxyphthalic anhydride, and2-(meth)acryloxycyclohexanedicarboxylic acid anhydride.

Ligand Monomer

Grafted to the primer layer is a ligand-functional alkenyl, preferably a(meth)acryloyl, (co)polymer layer. More specifically, theligand-functional (meth)acryloyl (co)polymer layer is grafted to thependent ethylenically unsaturated, polymerizable groups derived from theamine reactive monomer (such as those of Formula 2), which in turn iscovalently attached to the polyamine polymer by means of the reactionbetween the amine groups of the polyamine and the amine reactivefunctional group “A”.

The ligand-functional (meth)acryloyl (co)polymer comprises polymerizedmonomer units of the formula 3:

where

-   R¹ is H or C₁-C₄ alkyl;-   R² is a (hetero)hydrocarbyl group, preferably a divalent alkylene    having 1 to 20 carbon atoms;-   X¹ is —O— or —NR³—, where R³ is independently H or hydrocarbyl    preferably C₁-C₄ alkyl;-   R^(lig) is a ligand-containing group,-   p is 0 or 1; and-   m is 0 or 1.

The R^(lig) group may contain any functional group that will selectivelybind a biomacromolecule of interest, and may be selected fromtraditional ligands such as primary, secondary, tertiary, or quaternaryamine containing ligands; multifunctional ligands such as tyrosinol,tryptophanol, octopamine, 2-aminobenzimidazole,1,3-diamino-2-hydroxypropane, tris(2-aminoethyl)amine,diethylenetriamine, triethylenetetramine, tetraethylenepentamine, oragmatine containing ligands; and guanidine and biguanide groupcontaining ligands

In some embodiments, the ligand-functional alkenyl (co)polymer comprisespolymerized ligand-functional monomer units of the formula 4a or b:

wherein

-   R¹ is H or C₁-C₄ alkyl;-   R² is a (hetero)hydrocarbyl group, preferably a divalent alkylene    having 1 to 20 carbon atoms;-   each R³ is independently H or hydrocarbyl, preferably C₁-C₄ alkyl;-   R⁴ is H, C₁-C₄ alkyl or —N(R³)₂;-   R⁵ is H or hydrocarbyl, preferably C₁-C₄ alkyl or aryl;-   X¹ is —O— or —NR³—,-   o is 0 or 1, and-   n is 1 or 2.

Such ligand monomers may be made by condensation of an alkenyl oralkenoyl compound, typically a (meth)acryloyl halide, a(meth)acryloylisocyanate, or an alkenylazlactone, with a compound offormulas 5a or 5b:

where X¹, and R² to R⁴, and n are as previously defined.

Other ligand monomers may be made by condensation of a carbonylcontaining monomer, such as acrolein, vinylmethylketone, diacetoneacrylamide or acetoacetoxyethylmethacrylate, with a compound of formulas5a or 5b.

Preferably, the ligand-functional alkenyl (co)polymer layer alsocomprises units derived from a (meth)acryloyl monomer containing atleast two free radically polymerizable groups. Such multifunctional(meth)acryloyl monomer, including (meth)acrylate and (meth)acrylamidemonomers may be incorporated into the blend of polymerizable monomers toassist in branching or lightly crosslinking of the graftedligand-functional copolymer. Examples of useful multifunctional(meth)acrylates include, but are not limited to, di(meth)acrylates,tri(meth)acrylates, and tetra(meth)acrylates, such as ethyleneglycoldi(meth)acrylate, 1,6-hexanediol di(meth)acrylate, poly(ethylene glycol)di(meth)acrylates, polybutadiene di(meth)acrylate, polyurethanedi(meth)acrylates, and propoxylated glycerin tri(meth)acrylate,methylenebisacrylamide, ethylenebisacrylamide,hexamethylenebisacrylamide, diacryloylpiperazine, and mixtures thereof.

Surprisingly, it has been found that inclusion of such a multifunctional(meth)acrylate or (meth)acrylamide monomer typically increases thecapacity, particularly the dynamic binding capacity, of the graftedarticle for capturing biological species. Such comonomers are used inamounts of about 0.25% to about 5% by weight, preferably of about 1% toabout 3% by weight, relative to the total monomer weight. Higherconcentrations of polyfunctional comonomer often lead to decreasedcapacities. While not wanting to be bound by theory, it is believed thatthis comonomer promotes branching in the grafted (co)polymer layer,thereby leading to increased accessibility or availability of the ligandgroups.

It has been observed that the incorporation of a multifunctional(meth)acrylate or (meth)acrylamide monomer enables the preparation ofligand functionalized articles without the use of the primer layer.However, the primer layer enables a broader formulation window withinwhich one can maintain high functional capacity (static and dynamicbinding capacity) of the resultant articles.

The ligand-functional alkenyl (co)polymer layer (grafted to the primerlayer) may optionally comprise one or more hydrophilic monomers whichcomprise at least one alkenyl group, preferably a (meth)acryloyl group,and a hydrophilic group, including poly(oxyalkylene) and ionic groups,for providing hydrophilicity to the substrate, or for providing greaterselectivity to the substrate when binding biomaterials.

The hydrophilic ionic groups may be neutral, have a positive charge, anegative charge, or a combination thereof. With some suitable ionicmonomers, the ionic group can be neutral or charged depending on the pHconditions. This class of monomers is typically used to impart a desiredhydrophilicity to the porous base substrate in addition to the c)monomer. In applications for viral capture, the addition of a graftingionic monomer having a positive charge at the selected pH may allowselective binding of viruses while repelling positively chargedbiological materials such as antibodies.

In some preferred embodiments, the third monomer may have an acrylategroup, or other ethylenically unsaturated groups of reduced reactivity,and a poly(alkylene oxide) group; e.g. monoacrylated poly(alkylene oxidecompounds, where the terminus is a hydroxy group or an alkyl ethergroup.

In some embodiments the ionic monomers having a negative charge include(meth)acryloylsulfonic acids of Formula 6 or salts thereof

wherein, Y is a straight or branched alkylene (e.g., an alkylenes having1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms) and Lis —O— or —NR—, where R³ is H or C₁-C₄ alkyl-; Exemplary ionic monomersaccording to Formula 6 include, but are not limited to,N-acrylamidomethanesulfonic acid, 2-acrylamidoethanesulfonic acid,2-acrylamido-2-methyl-1-propanesulfonic acid, and2-methacrylamido-2-methyl-1-propanesulfonic acid. Salts of these acidicmonomers can also be used. Counter ions for the salts can be, forexample, ammonium ions, potassium ions, lithium ions, or sodium ions.

Other suitable ionic grafting monomers having a negative charge (at aselected pH) include sulfonic acids such as vinylsulfonic acid and4-styrenesulfonic acid; (meth)acrylamidophosphonic acids such as(meth)acrylamidoalkylphosphonic acids (e.g.,2-(meth)acrylamidoethylphosphonic acid and3-(meth)acrylamidopropylphosphonic acid; acrylic acid and methacrylicacid; and carboxyalkyl(meth)acrylates such as2-carboxyethyl(meth)acrylate, and 3-carboxypropyl(meth)acrylate. Stillother suitable acidic monomers include (meth)acryloylamino acids, suchas those described in U.S. Pat. No. 4,157,418 (Heilmann). Exemplary(meth)acryloylamino acids include, but are not limited to,N-acryloylglycine, N-acryloylaspartic acid, N-acryloyl-β-alanine, and2-acrylamidoglycolic acid. Salts of any of these acidic monomers canalso be used.

Some exemplary ionic grafting monomers that are capable of providing apositive charge (at a selected pH) are amino (meth)acrylates or amino(meth)acrylamides of Formula 7 or quaternary ammonium salts thereof. Thecounterions of the quaternary ammonium salts are often halides,sulfates, phosphates, nitrates, and the like.

where L is —O— or —NR³—, where R³ is H or C₁-C₄ alkyl-; and Y is analkylene (e.g., an alkylene having 1 to 10 carbon atoms, 1 to 6, or 1 to4 carbon atoms). Each R¹¹ is independently hydrogen, alkyl, hydroxyalkyl(i.e., an alkyl substituted with a hydroxy), or aminoalkyl (i.e., analkyl substituted with an amino). Alternatively, the two R¹¹ groupstaken together with the nitrogen atom to which they are attached canform a heterocyclic group that is aromatic, partially unsaturated (i.e.,unsaturated but not aromatic), or saturated, wherein the heterocyclicgroup can optionally be fused to a second ring that is aromatic (e.g.,benzene), partially unsaturated (e.g., cyclohexene), or saturated (e.g.,cyclohexane).

In some embodiments of Formula 7, both R¹¹ groups are hydrogen. In otherembodiments, one R¹¹ group is hydrogen and the other is an alkyl having1 to 10, 1 to 6, or 1 to 4 carbon atoms. In still other embodiments, atleast one of R¹¹ groups is a hydroxy alkyl or an amino alkyl that have 1to 10, 1 to 6, or 1 to 4 carbon atoms with the hydroxy or amino groupbeing positioned on any of the carbon atoms of the alkyl group. In yetother embodiments, the R¹¹ groups combine with the nitrogen atom towhich they are attached to form a heterocyclic group. The heterocyclicgroup includes at least one nitrogen atom and can contain otherheteroatoms such as oxygen or sulfur. Exemplary heterocyclic groupsinclude, but are not limited to imidazolyl. The heterocyclic group canbe fused to an additional ring such as a benzene, cyclohexene, orcyclohexane. Exemplary heterocyclic groups fused to an additional ringinclude, but are not limited to, benzoimidazolyl.

Exemplary amino acrylates (i.e., L in Formula 7 is —O—) includeN,N-dialkylaminoalkyl acrylates such as, for example,N,N-dimethylaminoethylacrylate, N,N-dimethylaminoethylmethacrylate,N,N-diethylaminoethyl acylate, N,N-diethylaminoethylmethacrylate,N,N-dimethylaminopropylacrylate, N,N-dimethylaminopropylmethacrylate,N-tert-butylaminopropylmethacrylate, N-tert-butylaminopropylacrylate andthe like.

Exemplary amino (meth)acrylamides, (i.e., L in Formula 7 is —NR³—)include, for example, N-(3-aminopropyl)methacrylamide,N-(3-aminopropyl)acrylamide, N-[3-(dimethylamino)propyl]methacrylamide,N-(3-imidazolylpropyl)methacrylamide, N-(3-imidazolylpropyl)acrylamide,N-(2-imidazolylethyl)methacrylamide,N-(1,1-dimethyl-3-imidazoylpropyl)methacrylamide,N-(1,1-dimethyl-3-imidazoylpropyl)acrylamide,N-(3-benzoimidazolylpropyl)acrylamide, andN-(3-benzoimidazolylpropyl)methacrylamide.

Exemplary quaternary salts of the ionic monomers of Formula 7 include,but are not limited to, (meth)acrylamidoalkyltrimethylammonium salts(e.g., 3-methacrylamidopropyltrimethylammonium chloride and3-acrylamidopropyltrimethylammonium chloride) and(meth)acryloxyalkyltrimethylammonium salts (e.g.,2-acryloxyethyltrimethylammonium chloride,2-methacryloxyethyltrimethylammonium chloride,3-methacryloxy-2-hydroxypropyltrimethylammonium chloride,3-acryloxy-2-hydroxypropyltrimethylammonium chloride, and2-acryloxyethyltrimethylammonium methyl sulfate).

Other monomers that can provide positively charged groups (at a selectedpH) to the base substrate include the dialkylaminoalkylamine adducts ofalkenylazlactones (e.g., 2-(diethylamino)ethylamine,(2-aminoethyl)trimethylammonium chloride, and3-(dimethylamino)propylamine adducts of vinyldimethylazlactone) anddiallylamine monomers (e.g., diallylammonium chloride anddiallyldimethylammonium chloride). Other examples include the quaternarysalt of dimethylaminoethyl methacrylate.

Neutral hydrophilic monomers that may be incorporated are poly(alkyleneoxide) monomers having a (meth)acryloyl or non-acryloyl ethylenicallyunsaturated group and a non-polymerizable terminus. Such monomers may beof the formula 8:CH₂═CR¹—C(O)—X¹—(CH(R¹)—CH₂—O)_(n)—R¹,  8wherein each R¹ is independently H or C₁-C₄ alkyl, X¹ is —O— or —NR³—,where R³ is H or C₁-C₄ alkyl and n is 2 to 100.

Others include the alkenylazlactones adducts of polyetheramines (such asthe monoamine, diamine and triamines based on the polyetheraminestructure). One example of these compounds is the Jeffamine® series,from Huntsman, The Woodlands, Tex., USA.

Such optional hydrophilic comonomers are used in amounts of about 1% toabout 70% by weight, preferably of about 5% to about 50% by weight,relative to the total monomer weight.

The ligand functional substrate may be prepared as shown in thefollowing Scheme 1. Here, a polyamino polymer (I) is crosslinked by thepolyfunctional crosslinking agent R⁸(Z)_(y) of Formula 1 to form thecrosslinked polymer (II). A portion of the unreacted amino groups of thepolyamino polymers are then functionalized with a (meth)acryloylcompound having an amine-reactive functional group of Formula 2 toproduce a crosslinked polyamino polymer having pendent ethylenicallyunsaturated groups (III). In the presence of a free-radical catalyst,polymerization is initiated in the presence of the ligand—functional(meth)acryloyl monomer of Formula 3 to produce an amino polymer havinggrafted ligand groups (IV) having “z” polymerized groups. Note withrespect to the following Schemes, the grafted polymer may haveadditional hydrophilic monomer units as described supra.

It will be understood with respect to Scheme 1 that the reactions of thepolyamine polymer with the polyfunctional crosslinking agent and withthe amine-reactive monomer are shown sequentially for clarity, but mayoccur essentially simultaneously (i.e., during the coating and dryingprocess). It will be further understood that the hydrophilic monomersand multifunctional acrylates are omitted from the Scheme for clarity,and the ligand-function monomer of Formula 3 is simplified for clarity.

In the reactions of Scheme 1, the ligand groups are directly graftedwith the ligand functional monomers. Alternatively, the crosslinkedpolyamino polymer may be indirectly functionalized with ligand groups,by grafting the intermediate III with a monomer having an alkenyl groupand a functional group capable of reacting with a ligand compound. Suchfunctionalized monomers are of the formula 9:

where

-   X¹ is —O— or —NR¹—, where R¹ is H or C₁-C₄ alkyl,-   R¹ is H or C₁-C₄ alkyl;-   R⁷ is a single bond or a (hetero)hydrocarbyl linking group,-   B is a nucleophilic or electrophilic functional group that is    reactive with ligand compound, such as those of Formulas 5a-b, and-   x is 0 or 1.

In some embodiments there is the proviso that when x is 0, then R⁷ is a(hetero)hydrocarbyl linking group. Generally when x is 0, then R⁷ is acovalent bond.

It will be understood that the functional monomers of formula 9 may beinclusive with those of Formula 2. Generally the “B” group will notreact readily with the amino groups of the primer layer. The Bfunctional group may further contain nucleophilic or electrophilicgroups, including halo, hydroxyl, amino (especially secondary amino),ketone (such as acetyl) carboxyl, aziridinyl, vinyloxy, and oxazolinyl,that are reactive toward, and will form a covalent bond with a ligandcompound for incorporating ligand groups into the article. In Scheme 2,the B* moiety is that group formed from the B functional group and theligand compound. For example, if B is an acetyl group, and the ligandcompound has an amine group, B* will be an imine group.

In some embodiments, the intermediate III of Scheme 2 is reacted with acarbonyl functional monomer, to provide a grafted copolymer havingpendent carbonyl groups, i.e. the B group of Formula 9 contains acarbonyl group. The grafted polymer comprises polymerized monomer unitsof an ethylenically unsaturated monomer having a carbonyl group,preferably a ketone or aldehyde group.

Generally, the carbonyl-functional (co)polymer comprises polymerizedmonomer units of the monomers selected from the group consisting of:acrolein, vinyl methyl ketone, vinyl ethyl ketone, vinyl isobutylketone, isopropenyl methyl ketone, vinyl phenyl ketone, diacetone(meth)acrylamide, acetonyl acrylate, and diacetone (meth)acrylate(co)polymers. Where B is a carbonyl group, and the ligand compound isattached with an amine group, B* will be an imine group.

The pendent carbonyl group then may be functionalized by nucleophilicgroups, typically an amine group to provide the requisite ligand group.For example the polymer of Scheme 2 having grafted carbonyl groups maybe subsequently functionalized with the guanidinyl compound of Formula5. This reaction typically requires the addition of an acid catalyst.

As with Scheme 1, the reactions are shown sequentially for clarity, butmay occur essentially simultaneously (i.e., during the coating anddrying process). It will be further understood that the hydrophilicmonomers and multifunctional acrylates are omitted from the Scheme forclarity.

By either reaction scheme, a primed substrate is provided with a graftedpolymer of the formula 10:-(M^(Lig))_(y)-(M^(Hydrophil))_(x)-(M^(crosslink))_(z)-,  10where

-   (M^(Hydrophil))_(x) are hydrophilic monomer units having “x”    polymerized monomer units,-   (M^(Lig))_(y) are ligand functional monomer units having “y”    polymerized monomer units,-   (M^(crosslink)) are multifunctional (meth)acrylate monomer units    having “z” polymerized monomer units,-   y is 10 to 100 wt. % of the monomer units;-   x is 0 to 90 wt. % of the monomer units;-   z is 0 to 5 wt. % of the monomer units.

It will be understood the monomer units are simplified for clarity. Itis believed that the multifunctional acrylate will crosslink the graftedligand functional polymer, and/or will further provide graftedhyperbranched polymers with ligand functional groups. It is furtherbelieved that free, ungrafted polymer may be present on the surface ofthe substrate, which may further be crosslinked and/or hyperbranched tothe extent that it is physically entangled with the grafted copolymer.

The substrate may be in any form such as particles, fibers, films orsheets. Suitable particles include, but are not limited to, organicparticles, inorganic particles, and porous and nonporous particles.Preferably the base substrate is porous. Suitable porous base substratesinclude, but are not limited to, porous particles, porous membranes,porous nonwoven webs, and porous fibers

The substrate may be formed from any suitable thermoplastic polymericmaterial. Suitable polymeric materials include, but are not limited to,polyolefins, poly(isoprenes), poly(butadienes), fluorinated polymers,chlorinated polymers, polyamides, polyimides, polyethers, poly(ethersulfones), poly(sulfones), poly(vinyl acetates), polyesters such aspoly(lactic acid), copolymers of vinyl acetate, such aspoly(ethylene)-co-poly(vinyl alcohol), poly(phosphazenes), poly(vinylesters), poly(vinyl ethers), poly(vinyl alcohols), and poly(carbonates).

In some embodiments, the thermoplastic polymer may be surface treated,such as by plasma discharge, to provide suitable functionality to thesurface of the substrate. Surface treatment provides functional groupssuch as hydroxyl groups that can improve wetting by the primer solution.One such useful plasma treatment is described in U.S. Pat. No. 7,125,603(David et al.).

Suitable polyolefins include, but are not limited to, poly(ethylene),poly(propylene), poly(1-butene), copolymers of ethylene and propylene,alpha olefin copolymers (such as copolymers of ethylene or propylenewith 1-butene, 1-hexene, 1-octene, and 1-decene),poly(ethylene-co-1-butene) and poly(ethylene-co-1-butene-co-1-hexene).

Suitable fluorinated polymers include, but are not limited to,poly(vinyl fluoride), poly(vinylidene fluoride), copolymers ofvinylidene fluoride (such as poly(vinylidenefluoride-co-hexafluoropropylene), and copolymers ofchlorotrifluoroethylene (such aspoly(ethylene-co-chlorotrifluoroethylene).

Suitable polyamides include, but are not limited to,poly(iminoadipolyliminohexamethylene),poly(iminoadipolyliminodecamethylene), and polycaprolactam. Suitablepolyimides include, but are not limited to, poly(pyromellitimide).

Suitable poly(ether sulfones) include, but are not limited to,poly(diphenylether sulfone) and poly(diphenylsulfone-co-diphenyleneoxide sulfone).

Suitable copolymers of vinyl acetate include, but are not limited to,poly(ethylene-co-vinyl acetate) and such copolymers in which at leastsome of the acetate groups have been hydrolyzed to afford variouspoly(vinyl alcohols).

A preferred substrate is a porous substrate that is a microporousmembrane such as a thermally-induced phase separation (TIPS) membrane.TIPS membranes are often prepared by forming a solution of athermoplastic material and a second material above the melting point ofthe thermoplastic material. Upon cooling, the thermoplastic materialcrystallizes and phase separates from the second material. Thecrystallized material is often stretched. The second material isoptionally removed either before or after stretching. Microporousmembranes are further disclosed in U.S. Pat. No. 4,529,256 (Shipman);U.S. Pat. No. 4,726,989 (Mrozinski); U.S. Pat. No. 4,867,881 (Kinzer);U.S. Pat. No. 5,120,594 (Mrozinski); U.S. Pat. No. 5,260,360(Mrozinski); and U.S. Pat. No. 5,962,544 (Waller, Jr.). Some exemplaryTIPS membranes comprise poly(vinylidene fluoride) (PVDF), polyolefinssuch as poly(ethylene) or poly(propylene), vinyl-containing polymers orcopolymers such as ethylene-vinyl alcohol copolymers andbutadiene-containing polymers or copolymers, and acrylate-containingpolymers or copolymers. For some applications, a TIPS membranecomprising PVDF is particularly desirable. TIPS membranes comprisingPVDF are further described in U.S. Pat. No. 7,338,692 (Smith et al.).

The substrate may be in any form such as films or sheets. Preferably thebase substrate is porous. Suitable porous base substrates include, butare not limited to, porous membranes, porous nonwoven webs, and porousfibers.

In many embodiments, the base substrate has an average pore size that istypically greater than about 0.2 micrometers in order to minimize sizeexclusion separations, minimize diffusion constraints and maximizesurface area and separation based on binding of a target molecule.Generally, the pore size is in the range of 0.1 to 10 micrometers,preferably 0.5 to 3 micrometers and most preferably 0.8 to 2 micrometerswhen used for binding of viruses. The efficiency of binding other targetmolecules may confer different optimal ranges.

In another exemplary embodiment the porous bases substrate comprises anylon microporous film or sheet, such as those described in U.S. Pat.No. 6,056,529 (Meyering et al.), U.S. Pat. No. 6,267,916 (Meyering etal.), U.S. Pat. No. 6,413,070 (Meyering et al.), U.S. Pat. No. 6,776,940(Meyering et al.), U.S. Pat. No. 3,876,738 (Marinacchio et al.), U.S.Pat. Nos. 3,928,517, 4,707,265 (Knight et al.), and U.S. Pat. No.5,458,782 (Hou et al.).

In other embodiments, the porous base substrate is a nonwoven web whichmay include nonwoven webs manufactured by any of the commonly knownprocesses for producing nonwoven webs. As used herein, the term“nonwoven web” refers to a fabric that has a structure of individualfibers or filaments which are randomly and/or unidirectionally interlaidin a mat-like fashion.

For example, the fibrous nonwoven web can be made by wet laid, carded,air laid, spunlaced, spunbonding or melt-blowing techniques orcombinations thereof. Spunbonded fibers are typically small diameterfibers that are formed by extruding molten thermoplastic polymer asfilaments from a plurality of fine, usually circular capillaries of aspinneret with the diameter of the extruded fibers being rapidlyreduced. Meltblown fibers are typically formed by extruding the moltenthermoplastic material through a plurality of fine, usually circular,die capillaries as molten threads or filaments into a high velocity,usually heated gas (e.g. air) stream which attenuates the filaments ofmolten thermoplastic material to reduce their diameter. Thereafter, themeltblown fibers are carried by the high velocity gas stream and aredeposited on a collecting surface to from a web of randomly dispersedmeltblown fibers. Any of the non-woven webs may be made from a singletype of fiber or two or more fibers that differ in the type ofthermoplastic polymer and/or thickness.

Further details on the manufacturing method of non-woven webs of thisinvention may be found in Wente, Superfine Thermoplastic Fibers, 48INDUS. ENG. CHEM. 1342 (1956), or in Wente et al., Manufacture OfSuperfine Organic Fibers, (Naval Research Laboratories Report No. 4364,1954).

In the process of forming the articles of the invention, the basesubstrate is first coated with the primer solution. For ease ofmanufacturing and reduced manufacturing costs, it is desirable to coat,crosslink, and functionalize the polyamine primer all in one operation.This one-step operation also has the advantage that the stoichiometry ofcrosslinking and functionalization can be readily controlled. Usefulcoating techniques include applying an aqueous solution or dispersion ofthe primer components onto the base substrate, followed by evaporatingthe solvent to form the primer coating. Coating methods include thetechniques commonly known as dip, spray, knife, bar, slot, slide, die,roll, or gravure coating.

Coating quality generally depends on mixture uniformity, the quality ofthe deposited liquid layer, and the process used to dry or cure theliquid layer. Coating quality of the primer layer is especiallyimportant since there are typically few or no chemical bonds between thesubstrate and the primer, and the primer layer is advantageous forsubsequent grafting of the ligand-functionalized polymer layer. Coatingquality may sometimes be improved by including alcohol cosolvents,especially with hydrophobic base substrates, as this improves thewetting of the substrate with the primer solution and thus ensures amore continuous coating. Improved crosslinking, leading to increaseddurability of the primer coating, and improved covalent attachment ofthe alkenyl group, leading to more efficient grafting, may sometimes beaccomplished by increasing the drying temperature.

Although the total concentration of reactants (polyamine polymer,crosslinking agent, and amine reactive monomer 2) in the primer solutionmay vary widely, it is generally desirable to keep that concentrationfairly low so as to minimize the thickness of the primer layer.Typically, the total concentration of reactants is from about 0.1% toabout 5% by weight, from about 0.25% to about 2% by weight, or fromabout 0.5% to about 1.5% by weight based on the total weight of theprimer solution. These concentrations will typically lead to about 0.5%to about 3% weight gain in the substrate upon priming.

In the second step of the process, the ligand-functionalized monomer isgraft (co)polymerized onto the alkenyl modified primer layer. Typically,the primed substrate is coated with an imbibing solution comprising theligand-functionalized monomer, any comonomers, an initiator, and asolvent for the mixture.

The polymerization may be initiated with either a thermal initiator orphotoinitiator, preferably a photoinitiator. Any conventional freeradical initiator may be used to generate the initial radical. Examplesof suitable thermal initiators include peroxides such as benzoylperoxide, dibenzoyl peroxide, dilauryl peroxide, cyclohexane peroxide,methyl ethyl ketone peroxide, hydroperoxides, e.g., tert-butylhydroperoxide and cumene hydroperoxide, dicyclohexyl peroxydicarbonate,2,2,-azo-bis(isobutyronitrile), and t-butyl perbenzoate. Examples ofcommercially available thermal initiators include initiators availablefrom DuPont Specialty Chemical (Wilmington, Del.) under the VAZO tradedesignation including VAZO™ 67 (2,2′-azo-bis(2-methylbutyronitrile))VAZO™ 64 (2,2′-azo-bis(isobutyronitrile)) and VAZO™ 52(2,2′-azo-bis(2,2-dimethylvaleronitrile)), and Lucidol™ 70 from ElfAtochem North America, Philadelphia, Pa.

Useful photoinitiators include benzoin ethers such as benzoin methylether and benzoin isopropyl ether; substituted acetophenones such as2,2-dimethoxyacetophenone, available as Irgacure™ 651 photoinitiator(Ciba Specialty Chemicals), 2,2 dimethoxy-2-phenyl-1-phenylethanone,available as Esacure™ KB-1 photoinitiator (Sartomer Co.; West Chester,Pa.), 1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-one,available as Irgacure™ 2959 (Ciba Specialty Chemicals), anddimethoxyhydroxyacetophenone; substituted α-ketols such as2-methyl-2-hydroxy propiophenone; aromatic sulfonyl chlorides such as2-naphthalene-sulfonyl chloride; and photoactive oximes such as1-phenyl-1,2-propanedione-2-(O-ethoxy-carbonyl)oxime. Particularlypreferred among these are the substituted acetophenones, and especially1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-one due toits water solubility. A particularly useful polymerizable photoinitiatoris one:one adduct of 2-vinyl-4,4-dimethylazlactone with Irgacure™ 2959,prepared as disclosed in Example 1 of U.S. Pat. No. 5,506,279 (Babu etal.), incorporated herein by reference

The initiator is used in an amount effective to facilitate free radicaladdition of the monomer(s) to pendent ethylenically unsaturated groupson the crosslinked polyamine polymer (derived from the amine reactivemonomer, component b)3)) and the amount will vary depending upon, e.g.,the type of initiator, and the molecular weight of the polymer and thedegree of functionalization desired. The initiators can be used inamounts from about 0.001 part by weight to about 5 parts by weight basedon 100 parts total monomer.

The solvent may be any polar solvent. In many embodiments the solvent iswater or a water/water-miscible organic solvent mixture. The ratio ofwater to organic solvent can vary widely, depending upon monomersolubility. With some monomers, it is typically greater than 1:1 (v/v)water to organic solvent, preferably greater than 5:1, and morepreferably greater than 7:1. With other monomers, a higher proportion oforganic solvent, even up to 100%, with some alcohols especially, may bepreferred.

Any such water miscible organic solvent preferably has no groups thatwould retard the polymerization. In some embodiments, the water misciblesolvents are protic group containing organic liquids such as the loweralcohols having 1 to 4 carbon atoms, lower glycols having 2 to 6 carbonatoms, and lower glycol ethers having 3 to 6 carbon atoms and 1 to 2ether linkages. In some embodiments higher glycols such as poly(ethyleneglycol) may be used. Specific examples are methanol, ethanol,isopropanol, n-butanol, t-butyl alcohol, ethylene glycol,methoxyethanol, ethoxyethanol, propoxyethanol, butoxyethanol, methylcarbitol, ethyl carbitol, and mixtures thereof.

In other embodiments, non-protic water miscible organic solvents thatcan also be used such as aliphatic esters and ketones and sulfoxidesmethoxyethyl acetate, ethoxyethyl acetate, propoxyethyl acetate,butoxyethyl acetate, triethyl phosphate, acetone, methyl ethyl ketone,methyl propyl ketone and dimethyl sulfoxide.

In embodiments where the polymer is functionalized in aqueous or otherprotic solvents, the “A” amine-reactive groups should be selected to besufficiently stable under the reaction conditions so the “A” groupsprimarily react with the amine groups of the polyamine.

The concentration of each component in the solution may vary dependingon a number of factors including, but not limited to, the graftingmonomer or monomers in the imbibing solution, the extent of graftingdesired, the reactivity of the grafting monomer(s), and the solventused. Typically, the total concentration of the monomers in the imbibingsolution ranges from about 0.1 wt % to about 60 wt %, desirably, fromabout 1 wt % to about 35 wt %, more desirably, from about 5% to about25%, based on a total weight of the solution. Following grafting,washing, and drying, typical total weight gains by the substrate are inthe range of about 5% to about 30%, in the range of about 10% to about25%, or in the range of about 12% to about 20%.

The method of grafting a ligand functionalized polymer to the surface ofthe substrate alters the original nature of the base substrate, as thesubstrate bears a grafted coating of the ligand functional polymer. Thepresent invention enables the formation of ligand functionalized polymersubstrates having many of the advantages of a base substrate (e.g.,mechanical and thermal stability, porosity), but with enhanced affinityfor biological species such as viruses, resulting from the monomers andsteps used to form a given functionalized substrate. While not wantingto be bound by theory, it is believed that the grafted, tentacularnature of the current coatings are responsible, at least in part, forthe improved binding capacities for biological species over materials inthe prior art.

The porous substrates having a coating of ligand-functionalized polymerare particularly suited as filter media, for the selective binding andremoval of target biological species including proteins, cells, celldebris, microbes, nucleic acids, and/or viruses from biological samples.The present disclosure further provides a method for the removal oftarget biological species from a biological sample by contacting thesample with the ligand polymer functionalized substrate as describedherein. As used herein “target biological species” may include acontaminant or a species of interest.

The ligand functionalized substrate is useful for the purification ofbiological or other fluid samples comprising biologically derivedspecies (biological species). Biological species include, but are notlimited to, cells, cell debris, proteins, nucleic acids, endotoxins, andviruses. Cells and cell debris include those derived from archaea,bacteria, and eucaryota. Bacteria include, but are not limited to,Gram-negatives such as Pseudomonas species, Escherichia coli,Helicobacter pylori, and Serratia marcesens; Gram-positives such asStaphylococcus species, Enterococcus species, Clostridium species,Bacillus species, and Lactobacillus species; bacteria that do not staintraditionally by Gram's method such as Mycobacterium species, andnon-vegetative forms of bacteria such as spores. Eucaryota include, butare not limited to, animal cells, algae, hybridoma cells, stem cells,cancer cells, plant cells, fungal hyphae, fungal spores, yeast cells,parasites, parasitic oocysts, insect cells, and helminthes. Proteins,include, but are not limited to, natural proteins, recombinant proteins,enzymes, and host cell proteins. Viruses include, but are not limitedto, enveloped species such as Herpesviruses, Poxviruses, Adenoviruses,Papovaviruses, Coronaviruses, retroviruses such as HIV, andPlasmaviridae; and non-enveloped species such as Caliciviridae,Corticoviridae, Myoviridae, and Picornaviridae.

In some embodiments, the biological species being removed from the fluidis the object of the purification. For example, a recombinant protein orenzyme may be prepared in cell culture or by fermentation, and thesubstrate can be used to capture the protein or enzyme as the first stepin the purification process. In another example, the substrate may beused to capture microorganisms from a fluid as the first step in aprocess of concentrating, enumerating, and/or identifying themicroorganisms.

In other embodiments, the biological species being removed from thefluid is a contaminant that must be removed prior to additionalprocessing steps for the fluid.

Significantly, many of the ligand functional substrates are useful underconditions of high salt concentration or high ionic strength, i.e., theyare “salt tolerant”. The term “salt” is meant to include all lowmolecular weight ionic species which contribute to the conductivity ofthe solution. The importance of utility of the ligand functionalsubstrates in the presence of salt is that many process solutions usedin biopharmaceutical or enzyme manufacture have conductivities in therange of 15-30 mS/cm (approximately 150-300 mM salt) or more. Salttolerance can be measured in comparison to that of the conventionalquaternary amine or Q ligand (e.g. trimethylammonium ligand), whoseprimarily electrostatic interactions with many biological speciesrapidly deteriorates at conductivities three- to six-fold less than thetarget range of 15-30 mS/cm. For example, membranes derivatized with theconventional Q ligand exhibit a drop in φX174 viral clearance from a sixlog-reduction value (LRV) to a one (1) LRV in going from 0 to 50 mM NaCl(ca. 5-6 mS/cm conductivity). Viruses such as φX174 which have pIs closeto 7 (are neutral or near-neutral at pH 7) are extremely difficult toremove from process streams. Similar problems are observed whenattempting to remove other biological species from process fluids. Forexample, when attempting to remove positively charged proteins such ashost cell proteins through the use of filtration devices functionalizedwith conventional Q ligands, the process fluid may have to be dilutedtwo-fold or more in order to reduce the conductivity to an acceptablerange. This is expensive and dramatically increases the overallprocessing time. Surprisingly, it has been found that ligandfunctionalized substrates in which the ligands comprise guanidine orbiguanide groups perform extremely well under high ionic strengthconditions.

The biological sample is contacted with the ligand functionalizedsubstrate for a time sufficient to interact and form a complex with thetarget biological species disposed (dissolved or suspended) in thesolution when the solution comprises from 0 to about 50 mM salt,preferably when the solution comprises from 0 to about 150 mM salt, morepreferably when the solution comprises from 0 to about 300 mM salt orhigher, such that the concentration of the target biological speciesremaining disposed in the solution is less than 50% of its originalconcentration. It is more preferred that the solution is contacted withthe ligand functionalized substrate for a time sufficient to interactand form a complex with the target biological species disposed in thesolution when the solution comprises from 0 to about 50 mM salt,preferably when the solution comprises from 0 to about 150 mM salt, morepreferably when the solution comprises from 0 to about 300 mM salt orhigher, such that the concentration of the target biological speciesremaining disposed in the solution is less than 10% of its originalconcentration. It is still more preferred that the solution is contactedwith the ligand functionalized substrate for a time sufficient tointeract and form a complex with the target biological species disposedin the solution when the solution comprises from 0 to about 50 mM salt,preferably when the solution comprises from 0 to about 150 mM salt, morepreferably when the solution comprises from 0 to about 300 mM salt orhigher, such that the concentration of the target biological speciesremaining disposed in the solution is less than 1% of its originalconcentration.

EXAMPLES

Test Methods

Static BSA Capacity Test for Membranes

Disks measuring 24 mm in diameter were punched out of the membranes andplaced in 5 mL centrifuge tubes. Bovine serum albumin solution (BSA;Catalog #A-7906; Sigma Aldrich; St. Louis Mo.) was prepared to aconcentration of about 3.0 mg/ml in 25 mM TRIS(tris(hydroxymethyl)aminomethane; Sigma Aldrich; St. Louis Mo.) buffer,pH 8.0 and 4.5 ml of the BSA solution was pipetted into each centrifugetube. The tubes were capped, and tumbled overnight (typically 14 hours).The supernatant solutions were analyzed using a UV-VIS spectrometer at279 nm (with background correction applied at 325 nm). The staticbinding capacities for the samples were determined by comparison to theabsorption of the starting BSA solution, and are reported in mg/mL asthe average of three replicates.

Dynamic BSA Capacity Test for Membranes:

The membranes were analyzed for binding of proteins by passing solutionsof the test analytes through a 6-layer stack of the membranes punchedout into 25-mm diameter disks and placed in a 25 mm diameter holderattached to an AKTA chromatography system (GE Healthcare, NY). Bovineserum albumin (BSA) was prepared at a concentration 1 mg/mL in 25 mMTRIS buffer, pH 8. The BSA feed solution was pumped through the membranestack at a flow rate of 1 mL/min and the UV absorbance of the effluentwas monitored at a wavelength of 280 nm. The dynamic binding capacity ofthe membrane was evaluated using standard chromatography techniques, andreported in mg/mL at 10% breakthrough.

Preparation of Primed Nylon Membrane

Polyethylenimine (PEI—MW 70,000, a 30% by weight aqueous solution,Cat#00618; Polysciences, Inc.; Warrington Pa.) was diluted to 1.0%solids with IPA (isopropanol). A 50 gram portion of this solution wasformulated with enough butanediol diglycidyl ether (BUDGE, SigmaAldrich) to react with 5% of the amine groups of the polymer and enoughglycidylmethacrylate (GMA, Sigma Aldrich) to react with 10% of the aminegroups. Primed membranes were prepared by dipping a 10 centimeter squarepiece of a nylon 66 membrane (single reinforced layer nylon three zonemembrane, nominal pore size 1.8 μm, #080ZN from 3M Purification, Inc.;Meriden Conn.), into the coating solution, removing excess coatingsolution with a #14 wire-wound coating rod, then allowing the membraneto air dry at ambient temperature for at least 15 minutes.

Examples 1-5, Comparative Examples C1-C2

Coating solutions (100 grams total weight) were prepared by dissolvingDAAm (diacetoneacrylamide; Alfa Aesar; Ward Hill Mass.) in the amountslisted in Table 1, 0.05 gram MBA (methylenebisacrylamide; SigmaAldrich), and 0.5 gram photoinitiator (Irgacure® 2959; Ciba; Basel,Switzerland) in deionized water. Sheets of primed nylon membrane weredip coated with the coating solutions of Examples 1-5 and sandwichedbetween two sheets of polyester film. The excess coating solution wasremoved using a #14 wire-wound coating rod and the sheets wereirradiated using a blacklight UV source for 15 minutes. The sheets wereturned over, and the opposite side was also exposed to the UV source for15 minutes. The polyester sheets were removed, and the grafted membraneswere placed in 500 mL polyethylene bottles. A 0.5 M aminoguanidinesolution was prepared by dissolving 6.15 grams of AG (Aminoguanidinesulfate; Alfa Aesar) in 100 mL of deionized water and adding 0.2 mL ofconcentrated hydrochloric acid, and added to each bottle. The membraneswere allowed to react with the aminoguanidine solution overnight, thenthe excess solution was poured off. The bottles were filled withdeionized water and shaken for 30 minutes to wash off residual monomeror ungrafted polymer. The water was replaced with 50 mM sodium acetate,pH 4.5, and washed for 30 minutes, washed another 30 minutes withdeionized water, and then allowed to dry. Samples were analyzed forstatic and dynamic BSA capacities (Table 1).

Comparative Example C1 was the primed membrane.

Comparative Example C2 was a membrane prepared similarly to Example 3but without reacting with aminoguanidine. Comparative Examples weretested for static BSA capacity.

TABLE 1 BSA Capacity DAAm (mg/mL) Example (grams) Static Dynamic 1 1 14NM 2 3 53 26 3 5 104 43 4 7 129 19 5 10 54  0 Comp. 1 0 25 NM Comp. 2 50 NM NM = Not Measured

Examples 6-9

Coating solutions were prepared at 5% total solids, according to theprocedure described above for Example 3, except that a co-monomer, DMA(Dimethylacrylamide, Sigma Aldrich), replaced a portion of the DAAm asshown in Table 2. Primed membranes were likewise coated, UV grafted,reacted with aminoguanidine, and washed, and tested for Static BSACapacity (Table 2).

TABLE 2 Static BSA Capacity Example DAAm (grams) DMA (grams) (mg/mL) 64.75 0.25 90 7 4.5 0.5 85 8 4.25 0.75 78 9 4.0 1.0 63

Examples 10-11

Coating solutions were prepared at 5% solids according to the proceduredescribed in Example 3. Example 10 was coated and grafted on a primedmembrane, as in Example 3, while Example 11 was coated and grafted on anunprimed membrane (#080ZN from 3M Purification, Inc). The graftedmembranes were reacted with aminoguanidine as in Example 3, except thatthe aminoguanidine sulfate solution was prepared at a 2.0 Mconcentration. Static BSA capacities were measured to be 97 and 88mg/mL, respectively, for Examples 10 and 11.

Examples 12-17 Preparation of4-(2-(methacryloyloxy)ethylaminocarbonylamino)butyl guanidiniumtriethylammonium sulfate (IEM-AGM triethylammonium sulfate)

Solution: A solution of agmatine sulfate (12.0 g, 53 mmol) dissolved in20 mL of deionized water was treated with triethylamine (7.31 mL, 53mmol) and 10 mL of methanol. The solution was then placed in a coldwater bath (approximately 15° C.) and 2-isocyanatoethyl methacrylate(7.50 mL, 53 mmol) was added dropwise. The solution was allowed to stirfor 90 min after which time NMR showed complete conversion to4-(2-(methacryloyloxy)ethylaminocarbonylamino)butyl guanidiniumtriethylammonium sulfate. The mixture was diluted to 100 grams total byaddition of methanol (46.5 grams). ¹H NMR (500 MHz, D₂O) δ 6.02 (s, 1H),5.61 (s, 1H), 4.11 (m, 2H), 3.33 (m, 2H), 3.06 (m, 2H), 3.04-2.98 (m,8H), 1.81 (s, 3H), 1.55-1.37 (m, 4H), 1.14 (t, J=7.3 Hz, 9 H).

Solid: A solution was prepared by dissolving 50 g of agmatine sulfate(50 g) in 500 mL of deionized water in a reaction flask followed bystirring in 200 mL of acetone and 40 mL triethylamine (287 mmol). Theflask was placed in a room temperature water bath while 40 mL of IEM(2-isocyanatoethyl methacrylate (283 mmol)) was slowly added by dropsover 5 minutes. The solution was stirred vigorously for 4 hours duringwhich a white precipitate formed. The precipitate was filtered out andacetone was removed from the filtered solution in a Rotovap. Theremaining solution was washed three times with ethyl acetate and oncewith methylene chloride. Both solvents caused phase separation and thesolvent phase was decanted. The remaining liquid was freeze dried toform 36 grams of white crystalline IEM-AGM. Multiple lots of IEM-AGMwere synthesized. ¹H NMR (500 MHz, CD₃OD) indicated differing levels ofpurity on a batch by batch basis: Lot A had a purity of 79%, Lot B had apurity of 87%, and Lot C had a purity of 90%. For Lots A and C, thestarting agmatine sulfate had a purity of 77 weight percent. Arecrystallized agmatine sulfate having a purity of about 80% was usedfor Lot B.

Six coating solutions, each having a total weight of 5 g, were preparedby dissolving 1.11 grams IEM-AGM Lot C (nominally 90% pure by NMR) and0.01 grams MBA (N,N′-Methylene bisacrylamide; Sigma Aldrich) indeionized water. A photoinitiator solution was prepared by dissolving5.0 grams Photoinitiator (IRGACURE 2959; BASF; Florham Park N.J.) inethanol to a total volume of 25 mL. Varying amounts of thephotoinitiator solution (Table 3) were delivered by micropipette to eachof the six coating solutions and mixed until homogeneous. Primedmembranes were placed on a sheet of polyester film, coated by pipettingapproximately 4.5 mL of each solution to the top surface of themembrane, and allowing to soak for about 2 minutes before a second sheetof polyester was placed on top of the membrane. Excess coating solutionwas removed using a #14 wire-wound coating rod, and the sandwich wasirradiated using a blacklight UV source for 15 minutes on each side asin Example 1. Grafted membranes were washed, dried, and evaluated forBSA binding capacities according to the procedure described in Example 1(Table 3).

TABLE 3 BSA Photoinitiator Capacity (mg/mL) Example (μL) Static Dynamic12 250 77 NM 13 125 91 NM 14 62.5 89 51 15 31.2 92 49 16 15.6 99 51 177.8 95 NM NM = Not Measured

Example 18

A coating solution was prepared as in Example 15, except that IEM-AGMused was Lot B (nominally 87% purity), and the solvent was 33/67deionized water/isopropanol by volume. A primed nylon membrane wascoated, grafted, washed, and dried according to the procedures describedin Example 1. The static BSA capacity was determined to be 98 mg/mL.

Examples 19-28

Coating solutions weighing a total of 5 grams were prepared bydissolving IEM-AGM (Lot 25A) and a co-monomer in the amounts listed inTable 4, and 1% by weight MBA based on the amount of IEM-AGM) in 50/50deionized water/isopropanol by volume, and adding 62.5 μL ofphotoinitiator solution prepared as described in Example 14. Theco-monomers were HEMA (Hydroxy ethyl methacrylate; Sigma Aldrich),MAPTAC (Methacrylamidopropyltrimethyl-ammonium chloride monomer—50% byweight in aqueous solution; TCI America; Portland Oreg.); AMPS-Na(Sodium salt of 2-acrylamido-2-methyl-1-propanesulfonic acid, 50 weight% solution—AMPS 2405; Lubrizol Corp.; Wickliffe Ohio); and DMA. Primedmembranes were coated, grafted, washed, and dried as in Example 1.Results are shown in Table 4.

TABLE 4 BSA Capacity IEM-AGM Co-monomer (mg/mL) Ex (grams) Co-monomer(grams) Static Dynamic 19 0.5 None 0 83 NM 20 0.55 HEMA 0.06 102 52 210.63 HEMA 0.13 126 68 22 0.71 HEMA 0.21 131 85 23 0.71 MAPTAC 0.21 81 3824 0.83 MAPTAC 0.33 101 54 25 0.63 AMPS-Na 0.13 116 68 26 0.71 AMPS-Na0.21 128 16 27 0.71 DMA 0.21 103 59 28 0.83 DMA 0.33 123  1 NM = NotMeasured

Examples 29-39

Coating solutions weighing a total of 5 grams were prepared bydissolving IEM-AGM Lot C (nominally 90% purity) and PEG400MA(Polyethyleneglycol 400 monomethacrylate co-monomer; Polysciences, Inc.)in the amounts listed in Table 5, and 1% MBA by weight, based on theamount of IEM-AGM, in 50/50 deionized water/isopropanol by volume, andadding 62.5 μL of the photoinitiator solution of Example 14. Primednylon membranes were coated, grafted, washed, dried and tested asdescribed in Example 1. Results are shown in Table 5.

TABLE 5 BSA Capacity IEM-AGM Comonomer (mg/mL) Example (grams) (grams)Static Dynamic 29 0.55 0.06 115 49 30 0.63 0.13 126 68 31 0.71 0.21 102100 32 0.71 0.29 130 108 33 0.71 0.36 129 94 34 0.71 0.43 116 94 35 0.710.50 106 72 36 0.83 0.25 114 98 37 0.83 0.33 130 107 38 0.83 0.41 121102 39 0.83 0.50 116 97

Examples 40-51

Coating solutions weighing a total of 5 grams were prepared bydissolving 0.71 grams IEM-AGM (Lot C) and a co-monomer in the amountslisted in Table 6, and 0.0071 gram MBA in 50/50 deionizedwater/isopropanol by volume, and adding 62.5 μL of the photoinitiatorsolution in Example 14. The co-monomers were PEG200MA(polyethyleneglycol 200 monomethacrylate; Polysciences, Inc.), and SR550 (polyethyleneglycol monomethylether monomethacrylate; Sartomer).Primed membranes were coated, grafted, washed, dried, and tested as inExample 1. Results are listed in Table 6.

TABLE 6 BSA Capacity Comonomer (mg/mL) Example Comonomer (grams) StaticDynamic 40 PEG200MA 0.07 132 87 41 PEG200MA 0.14 136 99 42 PEG200MA 0.21140 95 43 PEG200MA 0.29 144 98 44 PEG200MA 0.36 144 96 45 PEG200MA 0.43140 100 46 SR 550 0.14 126 65 47 SR 550 0.21 132 55 48 SR 550 0.29 12677 49 SR 550 0.36 132 68 50 SR 550 0.43 127 73 51 SR 550 0.50 125 73

Examples 52-60 Preparation of4-(2-(acryloylamino)-2-methylpropionylamino)butyl guanidiniumtriethylammonium sulfate (VDM-AGM triethylammonium sulfate)

Solid: A solution of agmatine sulfate (21.4 g, 94 mmol) dissolved in 100mL of deionized water was added to a stirred solution of triethylamine(13.9 mL, 100 mmol) in ethanol. The cloudy solution was treated withadditional water (10 mL) and became homogenous. The reaction mixture wasplaced in a water bath and 4,4-dimethyl-2-vinyl-1,3-oxazol-5(4H)-one(14.0 g, 101 mmol) was added dropwise over a five minute period. Afterstirring overnight the reaction mixture was concentrated under vacuum atambient temperature until the volume was approximately 50 mL. Themixture was then lyophilized to form 36 grams of VDM-AGM white powder.

Solution: A solution of agmatine sulfate (16.95 g, 74.3 mmol) dissolvedin 65 mL of deionized water was treated with triethylamine (7.50 g, 74.3mmol). The reaction mixture was placed in a water bath and4,4-dimethyl-2-vinyl-1,3-oxazol-5(4H)-one (10.56 g, 75.9 mmol) was addeddropwise over 10 minutes. The solution was allowed to stir for 90 minafter which time NMR showed complete conversion to4-(2-(acryloylamino)-2-methylpropionylamino)butyl guanidiniumtriethylammonium sulfate. ¹H NMR (500 MHz, D₂O) δ 6.20 (m, 1H), 6.06 (m,1H), 5.67 (d, 1H), 3.12-3.05 (m, 10H), 1.48-1.42 (m, 4H), 1.37 (s, 6H),1.18 (t, J=7.3 Hz, 9 H).

Coating solutions weighing 5 grams total were prepared by dissolvingsolid VDM-AGM, a comonomer, and MBA in the amounts listed in Table 7 indeionized water and adding 31.2 μL of the photoinitiator solution ofExample 14. Primed membranes were coated, grafted, washed, dried andtested as described in Example 1. Results are listed in Table 7. “Am” isacrylamide.

TABLE 7 Comono- BSA Capacity VDM-AGM MBA Comono- mer (mg/mL) Ex (grams)(grams) mer (grams) Static Dynamic 52 0.25 0.0025 None 0 21 NM 53 0.50.005 None 0 102 45 54 1.0 0.01 None 0 152 1 55 0.625 0.0062 AMPS-Na0.0312 120 55 56 0.625 0.0062 AMPS-Na 0.0625 96 46 57 0.5938 0.0062 DMA0.0312 155 0 58 0.5938 0.0062 Am 0.0312 171 0 59 0.5625 0.0062 DMA0.0625 181 0 60 0.5625 0.0062 Am 0.0625 192 0

The following Examples illustrate priming conditions for preparing anylon membrane suitable as a substrate for grafting.

Example 61

A priming solution was prepared by mixing 6.67 grams of PEI(Polyethylenimine; Polysciences, Inc.) with 6.67 grams of IPA to form a15% by weight solution. Vinyldimethylazlactone (VDM, 0.64 grams, enoughto react with 10% of the amine groups of the PEI) was added and mixed ona rocker. Within 15 minutes, the mixture had gelled, presumablyindicating that crosslinking occurred via Michael addition of aminegroups to the double bond of the acrylamide derived from VDM.

Example 62

A priming solution for a nylon membrane was prepared according to theprocedure of Example 61 except that the PEI solution was diluted to atotal of 40 grams with isopropanol (5% solids) before addition of theVDM. The reaction mixture was still fluid after 5 hours, but had gelledafter overnight reaction.

Example 63

A priming solution for a nylon membrane was prepared according to theprocedure of Example 62 except that 0.66 grams of GMA (Glycidylmethacrylate) was used instead of VDM. The reaction mixture was stillfluid after 18 hours reaction time.

Examples 64-67

For Example 64, a 1% solids by weight solution was prepared from bymixing 1.67 g PEI (30% solids by weight in aqueous solution) with IPA to50 grams total. A second 50 gram solution was prepared by dissolving14.7 mg butanediol diglycidylether (BUDGE), which was sufficient toreact with 2.5% of the amine groups of the PEI, in IPA solution. The twosolutions were mixed briefly to provide a 0.5% solids priming solution.Squares (10 cm) of a nylon 66 membrane (#080ZN; 3M Purification, Inc.)were dip coated with the priming solution and excess solution wasremoved using a #14 wire-wound coating rod. The coated membranes wereallowed to air dry at ambient temperature for at least 15 minutes. Someof the membranes were dip-coated a second time and allowed to air dry.Following drying, samples of each coated membrane were weighed todetermine the coating weight, and placed in 250 mL polyethylene bottlesfilled with deionized water. The bottles were sealed, and agitatedovernight to extract any unbound material. After drying, the membraneswere weighed to determine the amount of coating removed. Washed andunwashed membranes were tested for BSA capacity to determine theeffectiveness of the crosslinking (Table 8).

Examples 65, 66, and 67 were prepared in the same manner except that thepriming solutions had 5%, 10%, and 20% BUDGE, respectively (Table 8).

TABLE 8 Static BSA Capacity % Number (mg/mL) % Coating Ex Crosslinker ofCoats Unwashed Washed Removed 64 2.5 1 20 15 28 64 2.5 2 54 20 63 65 5 121 17 18 65 5 2 55 26 54 66 10 1 23 19 16 66 10 2 57 35 39 67 20 1 28 275 67 20 2 55 46 17

Examples 68-71

PEI Priming solutions were prepared at a final concentration of 1%solids with the amounts of crosslinker in Table 9, and membranes werecoated as described in Example 64. Some samples were air-dried as inExample 4, and others were dried in an oven at 45-50° C. for 2 hours.All of the samples were washed with deionized water as in Example 64,and measured for static BSA capacity (Table 9).

TABLE 9 Static BSA Capacity (mg/mL) Example % Crosslinker Air-dried Ovendried 68 2.5 16 29 69 5 21 22 70 10 24 14 71 20 28 10

Examples 72-75

A primed nylon membrane for Example 72 was prepared according to theprocedure described for “Preparation of primed nylon membrane”. Example73-75 were prepared in the same manner with the following exceptions:Example 73 had no GMA; Example 74 was prepared with VDM as a graft sitemonomer instead of GMA; and Example 75 was prepared with deionized wateras the solvent for primer solution. For each example, an additionalprimed membrane was washed with acetate buffer (50 mM sodium acetate, 40mM sodium chloride, pH 4.5) for 1 hour, followed by a deionized waterwash for 1 hour, and then allowed to air-dry. All of the membranes weregrafted according as described in Example 32, except that the coatingsolvent was methanol, and tested for static and dynamic BSA capacities(Table 10).

TABLE 10 Static BSA Capacity (mg/mL) Dynamic BSA Ex Wash Primed OnlyGrafted Capacity (mg/mL) 72 No 59 126 97 Yes 25 127 92 73 No 62 115 64Yes 22 126 33 74 No 18 90 70 Yes 20 93 71 75 No 19 104 82 Yes 19 105 83

Examples 76-79

In Examples 76-79 nylon membranes were prepared and grafted according tothe procedure described in Example 72 except that the weight percent(based on the weight of the IEM-AGM monomer) of MBA in the coatingsolution was varied (Table 11).

Examples 80-84 were prepared in the same manner except that unprimednylon membranes (#080ZN; 3M Purification, Inc.) were grafted and tested(Table 11).

TABLE 11 BSA Capacity (mg/mL) Example % MBA Static Dynamic 76 0.5 102114 77 1 112 114 78 2 132 105 79 3 132 78 80 0.5 100 2 81 1 111 28 82 2119 99 83 3 100 68 84 4 80 57

Examples 85-87

A coating solution was prepared from 17.0 grams of IEM-AGM (assayed tobe 58.8% pure by NMR, 10.0 grams actual ligand monomer) and 0.5 gramsVAZPIA (prepared as disclosed in Example 1 of U.S. Pat. No. 5,506,279),dissolved in 2 grams of methanol, and diluted to 50 grams total withdeionized water. Portions of this solution were formulated for graftingby adding 0, 1, or 2% of MBA based on the weight of ligand monomer forExamples 85, 85, and 87, respectively. The nylon membranes wereUV-grafted according to the procedure described in Example 1, using anirradiation time of 45 minutes per side, washed, dried, and analyzed forBSA capacities (Table 12).

TABLE 12 BSA Capacity (mg/mL) Example % MBA Static Dynamic 85 0 93 49 861 95 73 87 2 92 88

Examples 88-93

Nylon membranes were prepared with the priming solution as describedabove in Example 72 except that 5% GMA based on amount of amine groupsin the PEI was used for Example 88, 10% GMA for Example 89, and 15% GMAfor Example 90. Similarly, membranes were primed using 5%, 10%, and 15%VDM to provide the graft sites, Examples 91-93, respectively. Themembranes were all grafted with diacetone acrylamide and functionalizedaccording to the procedure described in Example 3 and evaluated forstatic BSA capacity (Table 13).

TABLE 13 Graft Site Component Static BSA Capacity Example Monomer Weight% (mg/mL) 88 GMA 5 93 89 GMA 10 91 90 GMA 15 93 91 VDM 5 102 92 VDM 1094 93 VDM 15 90

Examples 94-98

Coating solutions weighing a total of 5 grams each were prepared bydissolving IEM-AGM (nominally 96% pure by NMR) and PEG400MA in theamounts listed in Table 14, and 1% MBA by weight based on the amount ofIEM-AGM, in methanol, and adding 31.2 μL of the photoinitiator solutionof Example 14. Primed membranes were coated, grafted, washed and driedas described in Example 1. Following the measurement of dynamic bindingcapacity as described in the Test Methods section, any bound protein waseluted by washing with 1 M NaCl solution, re-equilibrated with 25 mMTRIS buffer containing 50 mM NaCl, pH 8. The dynamic capacity test wasthen repeated using a 1 mg/mL BSA solution in the same TRIS/salt buffer.Dynamic capacity results are listed in the Table as “Dynamic (salt)”.These Examples illustrate that these highly charged membranes sometimesperform better in higher conductivity media.

TABLE 14 BSA Capacity (mg/mL) IEM-AGM PEG400MA Dynamic Example (grams)(grams) Static Dynamic (salt) 94 0.60 0.24 119 82 90 95 0.65 0.26 121 23100 96 0.70 0.28 120 8 111 97 0.75 0.30 117 11 108 98 0.80 0.32 106 2117

Examples 99-101 Synthesis of4-(2-(methacryloyloxy)ethylaminocarbonylamino)butyl guanidinium sodiumsulfate (IEM-AGM sodium sulfate)

Agmatine sulfate (100 g, 397 mmol) was dissolved in 400 mL of aqueous1.00 N NaOH. Acetone (200 mL) was then added and the stirred mixture wascooled to about 10° C. in a cold water bath. An additional 80 mL of H₂Owas added to keep the agmatine sulfate in solution. 2-isocyanatoethylmethacrylate (58.0 mL, 411 mmol) was then added to the reaction mixture,via an addition funnel, over a period of 30 min. After stirring anadditional 45 min, the reaction mixture was placed on a rotaryevaporator at ambient temperature. After pulling off most of theacetone, the reaction mixture was transferred to a separatory funnel andwashed with ethyl acetate (2×250 mL) and methylene chloride (2×200 mL).The remaining aqueous solution was adjusted to pH 7 by addition of asmall amount of dilute sulfuric acid and then placed on a rotaryevaporator at ambient temperature to draw off any remaining volatiles.Lyophilization yielded the title compound (162 g) as a white powder. ¹HNMR (500 MHz, D₂O) δ 6.14 (s, 1H), 5.73 (s, 1H), 4.23 (t, J=5.2 Hz, 2H),3.45 (t, J=5.4 Hz, 2H), 3.18 (t, J=7.0 Hz, 2H), 3.12 (t, J=6.4 Hz, 2H),1.22 (s, 3H), 1.61-1.48 (m, 4H).

Synthesis of 4-(2-(methacryloyloxy)ethylaminocarbonylamino)butylguanidinium hemisulfate (IEM-AGM hemisulfate)

A solution of agmatine hemisulfate (2.00 g, 11.2 mmol) dissolved in 20mL of ethanol was treated with 2-isocyanatoethyl methacrylate (1.50 mL,10.6 mmol) over the course of a few minutes. The solution was allowed tostir for 30 min after which time the reaction mixture was concentratedat ambient temperature. The resulting syrup was concentrated twice withtoluene to give the title compound as a white foam. ¹H NMR (500 MHz,D₂O) δ 6.13 (s, 1H), 5.73 (s, 1H), 4.23 (t, J=4.8 Hz, 2H), 3.44 (t,J=4.8 Hz, 2H), 3.18 (t, J=7.0 Hz, 2H), 3.11 (t, J=6.7 Hz, 2H), 1.92 (s,3H), 1.65-1.49 (m, 4H).

Coating solutions weighing a total of 5 grams each were prepared bydissolving IEM-AGM (counterion noted in Table 15) and PEG400MA in theamounts listed in Table 15, and 1% MBA by weight based on the amount ofIEM-AGM, in methanol, and adding 31.2 μL of the photoinitiator solutionof Example 14. Primed membranes were coated, grafted, washed and driedas described in Example 1. Dynamic capacities were measured using BSAdissolved in the TRIS/NaCl buffer described in Example 94.

TABLE 15 IEM- BSA Capacity AGM PEG400MA (mg/mL) Example Counterion(grams) (grams) Static Dynamic 99 sodium sulfate 0.50 0.20 131 91 100sodium sulfate 0.67 0.27 116 112 101 hemisulfate 0.55 0.22 130 106

Example 102 Synthesis of6-(2-(acryloylamino)-2-methylpropionylamino)hexyl guanidinium sodiumsulfate

Aminohexylguanidine sulfate (32.4 g, 127 mmol) was dissolved in 120 mLof aqueous 1.00 N NaOH. Acetone (60 mL) was then added and the stirredmixture was cooled to about 10° C. in a cold water bath. An additional60 mL of H₂O was added to keep the aminohexylguanidine sulfate insolution. 4,4-dimethyl-2-vinyl-1,3-oxazol-5(4H)-one (17.6 g, 137 mmol)was then added to the reaction mixture, via an addition funnel, over aperiod of 10 min. After stirring an additional 60 min, the reactionmixture was placed on a rotary evaporator at ambient temperature. Afterpulling off most of the acetone, the reaction mixture was transferred toa separatory funnel and washed with ethyl acetate (2×75 mL) andmethylene chloride (2×25 mL). The remaining aqueous solution was broughtto pH 7 by addition of dilute sulfuric acid and then placed on a rotaryevaporator at ambient temperature to draw off any remaining volatiles.Lyophilization yielded the title compound (49 g) as a white powder ¹HNMR (500 MHz, D₂O) δ 6.37 (m, 1H), 6.25 (m, 1H), 5.84 (m, 1H), 3.26-3.24(m, 4H), 1.68-1.38 (m, 14H). This monomer (0.71 grams), MBA (0.07grams), PEG400MA (0.28 grams), and photoinitiator (31.2 μL of thephotoinitiator solution of Example 14) were dissolved in enough methanolto provide 5 grams of coating solution. Primed membranes were coated,grafted, washed and dried as described in Example 1. Static and dynamiccapacities for the grafted membrane were 110 and 71 mg/mL, respectively.

Example 103

HCP and DNA removal—Salt Tolerance

Membranes prepared according to the recipe of Example 96 were analyzedfor binding of HCP (host cell proteins) and DNA by passing diafilteredCHO cell culture solutions through a 6-layer stack of the membranespunched out into 25-mm diameter disks and placed in a 25 mm diameterholder attached to an AKTA chromatography system (GE Healthcare, NY).The challenge solution was prepared by first filtering CHO cell culturewith a 0.2 um filter then concentrating and diafiltering the solutioninto 25 mM TRIS buffer, pH 8. The diafiltration was performed on aMillipore Labscale TFF System with a Pellicon Biomax 50 cassette using 8volumes of TRIS. The resulting diafiltered material had a HCPconcentration of 225000 ng/mL, DNA concentration of 75000 ng/mL and aconductivity of 1.5 mS/cm. To demonstrate salt tolerance, solutions ofthis material with higher salt concentrations were prepared by adding5.0 M NaCl to reach conductivity levels of 5, 10, 20, 25, 30, and 40mS/cm. The resulting challenge solutions were pumped through themembrane stack at a flow rate of 3 mL/min and the FT pool was collectedwith a fraction collector Frac-950 (GE Healthcare, NY). The HCPconcentration of each pool was measured with a Chinese Hamster OvaryHost Cell Protein—3^(rd) Generation ELISA kit (Cygnus Technologies, Inc)following the standard procedure. DNA concentrations were measure with aQuant-iT PicoGreen dsDNA Assay Kit (Invitrogen). Results are listed inTable 16 as log reduction values (LRV) relative to the startingsolution.

TABLE 16 Conductivity DNA HCP (mS/cm) (LRV) (LRV) 1.5 2.2 1.7 5 2 2 102.5 1.7 20 3.1 1.7 25 2.9 1.2 30 2 0.9 40 1.9 0.8

Example 104

A coating solution was prepared from 10 grams ofN-(3-dimethylaminopropyl)-acrylamide, 0.1 grams of MBA, 0.5 grams ofIrgacure 2959, and 90 grams of deionized water. A primed membrane wascoated and UV-grafted with this solution according to the proceduredescribed in Example 1, washed, and dried. When assayed for static BSAcapacity, this membrane absorbed all of the BSA from solution,indicating a BSA capacity of >195 mg/mL.

The invention claimed is:
 1. A ligand functional substrate comprising:a) a substrate b) a primer layer disposed on the substrate comprising: acrosslinked polyamine polymer having ethylenically unsaturatedpolymerizable groups; c) a ligand-functional alkenyl (co)polymer layergrafted to the ethylenically unsaturated polymerizable group of theprimer layer.
 2. The ligand-functional substrate of claim 1, wherein thecrosslinked polyamine polymer having ethylenically unsaturatedpolymerizable groups is the reaction product of: 1) a polyamine polymer2) a polyfunctional crosslinking agent for the polyamine polymer; and 3)a monomer having an amine-reactive functional group and an ethylenicallyunsaturated polymerizable group.
 3. The ligand-functional substrate ofclaim 1, wherein said ligand is selected from the group consisting oftyrosinol, tryptophanol, octopamine, 2-aminobenzimidazole,1,3-diamino-2-hydroxypropane, tris(2-aminoethyl)amine,diethylenetriamine, triethylenetetramine, tetraethylenepentamine,agmatine, guanidine and biguanide ligands.
 4. The ligand-functionalsubstrate of claim 1, wherein the ligand-functional alkenyl (co)polymercomprises polymerized monomer units of the formula:

wherein R¹ is H or C₁-C₄ alkyl; R² is a (hetero)hydrocarbyl group; X¹ is—O—or —NR³—, where R³ is independently H or C₁-C₄ alkyl; R^(lig) is aligand-containing group, p is 0 or 1; and m is 0 or
 1. 5. Theligand-functional substrate of claim 1, wherein the ligand-functionalalkenyl (co)polymer comprises polymerized monomer units of the formula:

wherein R¹ is H or C₁-C₄ alkyl; R² is a (hetero)hydrocarbyl group; eachR³ is independently H or hydrocarbyl; R⁴ is H, C₁-C4 alkyl or —N(R³)2;R⁵ is H or hydrocarbyl; X¹ is —O—or —NR³—, o is 0 or 1, and n is 1 or 2.6. The ligand-functional substrate of claim 1, wherein theligand-functional alkenyl (co)polymer further comprises polymerizedpolar monomer units.
 7. The ligand-functional substrate of claim 6,wherein the ligand-functional alkenyl (co)polymer further comprisespolymerized cationic or anionic monomer units.
 8. The ligand-functionalsubstrate of claim 6, wherein the polar monomer units comprise thepoly(oxyalkylene) (meth)acrylate monomer units.
 9. The ligand-functionalsubstrate of claim 8, wherein the poly(oxyalkylene) (meth)acrylatemonomer units are of the formula: CH₂═CR¹—C(O)—X¹—(CH(R¹)—CH2—O)_(n)—R¹,wherein each R¹ is independently H or C₁-C₄ alkyl, X¹ is —O—or —NR³—,where R³ is H or C₁-C₄ alkyl and n is 2 to
 100. 10. Theligand-functional substrate of claim 2 wherein the polyamine polymer isselected from the group consisting of polyethylenimine, polylysine,polyaminoamides, polydimethylamine-epichlorohydrin-ethylenediamine,polyaminosiloxanes and dendrimers formed from polyamidoamine andpolypropylenimine.
 11. The ligand-functional substrate of claim 1wherein said primer layer comprises 0.1 to 5 wt. % of the substrate. 12.The ligand-functional substrate of claim 1 wherein saidligand-functional alkenyl (co)polymer comprises 5 to 30 wt.% of theligand functional substrate.
 13. The ligand-functional substrate ofclaim 1 wherein said ligand groups are of the formula:

wherein each R³ is independently H or C₁-C₄ alkyl; R⁴ is H, C₁-C₄ alkylor —N(R³)₂; and n is 1 or
 2. 14. The ligand-functionalized substrate ofclaim 1 comprising a ligand-functional alkenyl (co)polymer layer of theformula:-(M^(Lig))_(y)-(M^(Hydrophil))_(x)-(M^(crosslink))_(z) -, wherein(M^(Hydrophil))_(x) are hydrophilic monomer units having “x” polymerizedmonomer units, (M^(Lig))_(y) are ligand functional monomer units having“y” polymerized monomer units, (M^(crosslink))are multifunctional(meth)acryloyl monomer units having “z” polymerized monomer units, y is10 to 100 wt. % of the monomer units; x is 0 to 90 wt. % of the monomerunits and; z is 0 to 5 wt. % of the monomer units, based on 100 wt. %total monomers.
 15. The ligand-functionalized substrate of claim 2wherein the polyfunctional crosslinking agent is of the formula:R⁸—(Z)_(y), where R⁸ is a (hetero)hydrocarbyl group, Z is anamine-reactive group, and y is ≧2.
 16. The ligand-functionalizedsubstrate of claim 2 wherein said polyfunctional crosslinking agent ofthe formula:

where R⁶ is an alkylene or arylene having a valence of n; X¹ is —O—or—NR¹ 13 , where R¹is H or C₁-C₄ alkyl, and n is 2 to
 6. 17. Theligand-functionalized substrate of claim 2 wherein said monomer havingan amine-reactive functional group is of the formula:

wherein X¹ is —O—or —NR¹—, where R¹ is H or C₁-C₄ alkyl, R¹ is H orC₁-C₄ alkyl; R⁷ is a single bond or a hydrocarbyl linking group, A is afunctional group that is reactive with the amino groups of the polyaminepolymer, and x is 0 or
 1. 18. The ligand-functionalized substrate ofclaim 15 wherein the crosslinking agent for the polyamine polymer isused in amounts wherein the number of equivalents of amine reactivegroups Z is at least 2% to 20%, relative to the equivalent of aminegroups in the polyamine polymer.